The objectives of the proposed research program are to purify the inhibitors of insulin protease present in human plasma to homogeneity, to examine the reactive site(s) for the amino acid(s) therein present, to examine also their ability to inhibit other proteolytic enzymes and to determine the amino acid sequence. The methods to be used for purification of the inhibitors include the following: (1) Separation and purification to homogeneity in the presence of various agents which favor dissociation of enzyme-inhibitor complexes (7M urea, strongly acid pH, etc.) on Sephadex G-50 followed by (2) ion-exchange chromatography, (QAE and S P Sephadexes, etc.). Protein modifying techniques such as treatment with maleic anhydride or butanedione will be used to determine if lys or arg residues are necessary for inhibitory activity. Prolonged incubation with pure enzyme and inhibitor will demonstrate (if inhibitory activity disappears with time) that the enzyme cleaves peptide bonds in the inhibitor. The ability of the inhibitors to block the action of other proteolytic enzymes such as trypsin and chymotrypsin, will be tested using synthetic substrates. The amino acid sequence will be studied cleaving the inhibitors first with trypsin and chymotrypsin to obtain overlapping peptides which will then be purified by ion-exchange chromatography.